Computational protocol: Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing

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Protocol publication

[…] Isolates were stored on beads at −80°C until processed. The cultures were recultured by the Infection Group, School of Medicine, University of St Andrews. DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The quality of the DNA was measured as A280 nm/A260 nm ratio on NanoVue (GE Healthcare, Little Chalfont, UK), and the concentration of double-stranded DNA was assessed using dsDNA BR Kit on a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). One-nanogram samples of DNA were used to construct the libraries with Nextera XT kit (Illumina Inc, San Diego, CA, USA). The normalized libraries were sequenced using a 2 × 250 pair-end read of a 500-cycle v2 kit on a MiSeq platform (Illumina Inc, San Diego, CA, USA) using a resequencing workflow. The Illumina sequences generated were deposited in the European Nucleotide Archive under the study accession number ERP023446. Using SMALT (Wellcome Trust Sanger Institute; www.sanger.ac.uk/resources/software/smalt/), reads were initially mapped to the chromosome of P. aeruginosa PAO1 (accession number AE004091), and single nucleotide polymorphisms (SNPs) were identified as described previously . In addition, the chromosomes of a representative selection of P. aeruginosa strains – B136-33 (accession number CP004061), DK2 (CP003149), LES431 (CP006937), LESB58 (FM209186), M18 (CP002496), MTB-1 (CP006853), NCGM2.S1 (AP012280), PA1 (CP004054), PA38182 (HG530068), RP73 (CP006245), SCV20265 (CP006931), UCBPP-PA14 (CP000438), VRFPA04 (CP008739) and YL84 (CP007147) – were used to provide a wider context for the hospital isolates. For each of these additional P. aeruginosa strains, artificial 250bp pair-end reads fastq files were generated using a python script. The generated fastq files were mapped along with the outbreak isolates to the chromosome of P. aeruginosa PAO1 and SNPs. Recombination was detected in the genomes using Gubbins (http://sanger-pathogens.github.io/gubbins/) .The core genome regions of the PAO1 and UCBPP-PA14 chromosome were defined by human curation using pairwise Blast comparisons with each other and other P. aeruginosa strains . The Artemis Comparison Tool was used to visualize the comparisons . SNPs falling inside mobile genetic elements were excluded from the core genome, as well as those falling in regions predicted by Gubbins to have occurred by recombination. Phylogenetic trees were constructed separately using RAxML v7.0.4 for all sites in the core genomes containing SNPs, using a general time reversible model with a gamma correction for among-site rate variation , . For a higher resolution phylogeny, ICU isolates that clustered on a branch with UCBPP-PA14 (CP000438) were mapped to this sequence as described above. […]

Pipeline specifications

Software tools SMALT, Gubbins, ACT, RAxML
Databases ENA
Applications Phylogenetics, WGS analysis
Organisms Pseudomonas aeruginosa, Homo sapiens